Method of imaging colorectal carcinoma lesion and composition for use therein

ABSTRACT

This invention provides a method of imaging a colorectal carcinoma lesion in a human patient which comprises administering to the patient a monoclonal antibody capable of binding to a cell surface antigen associated with the coloretal carcinoma lesion and which is labeled with an imaging agent under conditions so as to form a complex between the monoclonal antibody and the cell surface antigen, imaging any complex so formed, and thereby imaging the colorectal carcinoma lesion. 
     This invention also provides a monoclonal antibody designated AS 33 (ATCC Accession No. HB 8779) and the hybridoma which produces it.

This is a division of application Ser. No. 327,765, filed Mar. 23, 1989now abandoned and is a continuation in part of U.S. Ser. No. 118,411 nowabandoned, filed Nov. 6, 1987, which is a continuation of U.S. Ser. No.724,991, filed Apr. 19, 1985 (now abandoned), the contents both of whichare hereby incorporated by reference into the present application.

Throughout this application various publications are referenced bynumbers within parentheses. Full citations for these publications may befound at the end of the specification immediately preceding the claims.The disclosures of these publications, in their entireties, are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

In 1987, it is estimated that 145,000 cases of colon carcinoma will bediagnosed in the United States (1). Due to lack of effective systemictherapy for metastatic disease, 47% of all patients with colon cancer,and the majority of those with metastatic disease succumb to theirmalignancies (1). Thus far, chemotherapeutic agents have not exhibitedsufficient antitumor activity to prolong the survival of patients withmetastatic disease. Development of new diagnostic and therapeuticmodalities is therefore of utmost importance.

The use of radioisotope labeled monoclonal antibodies as a diagnostictool has been reported. Of the numerous monoclonal antibody studieswhich have been reported, the parameters defining specific tissuelocalization with radiolabeled monoclonal antibodies remain obscure.Detection of radioactivity over tumor sites by external scintigraphydoes not always indicate specific localization (7). Mouse imagingexperiments demonstrate that tissue vascularity and blood clearance ofradiolabeled antibody strongly affect tissue antibody content (7). In aprevious clinical trial of one antibody (8), tumor/nontumorradioactivity ratios were affected by several parameters including thetime interval between antibody administration and biopsy as well as thetypes of normal tissue biopsied. Another issue is the antigenspecificity of antibody localization. Isotype matched control antibodiesare important to definitively prove that localization is specific.

These issues are addressed in this study by: 1) tissue biopsies from allpatients seven days after administration of labeled antibody to documenttumor and measure antibody uptake in tumor and normal (liver) tissues;2) ^(99m) Tc HSA scans were performed in all patients to show thatradioactivity in tumors was not due to vascularity of tumors (blood flowstudies); and 3) a control antibody was compared to AS 33 to demonstratethat ¹³¹ I-AS 33 localization was specific.

SUMMARY OF THE INVENTION

This invention provides a method of imaging a colorectal carcinomalesion in a human patient which comprises administering to the patient amonoclonal antibody capable of binding to a cell surface antigenassociated with the colorectal carcinoma lesion and which is labeledwith an imaging agent under conditions so as to form a complex betweenthe monoclonal antibody and the cell surface antigen, imaging anycomplex so formed, and thereby imaging the colorectal carcinoma lesion.

This invention also provides a monoclonal antibody designated AS 33(ATCC Accession No. HB 8779) and the hybridoma which produces it.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 (A-C) show the reactivity of monoclonal antibody AS 33 with apanel of tumor cell lines.

FIGS. 2 (A) and (B) illustrate the reactivity of monoclonal antibody AS33 with frozen sections of normal human fetal and adult tissues.

FIGS. 3(A) and 3(B) summarizes tissue and blood dosimetry results fortwelve patients who were administered radiolabeled AS 33.

FIG. 4 shows gamma camera imaging results at 4 h, day 1, day 4 and day 6using ¹³¹ I labeled AS 33. At 24 hrs a primary colon cancer can beidentified in the right lower quadrant of the abdomen. As blood born ¹³¹I antibody is cleared (6 d) liver metastasis and lymph mode metastasisare seen. All these sites of disease were confirmed at surgery andbiopsies demonstrated antibody localization to these lesions and thatthese lesions were colon carcinomas. Arrows indicates lesion sites.

FIG. 5 shows gamma camera imaging results at day 4 and day 6 using ¹³¹ Ilabeled AS 33. A ^(99m) TcHSA scan is shown in left panel whichdemonstrates a hypovascular lesion in the right lobe of the liver. ¹³¹I-AS 33 scans on day 4 and 6 show antibody localization in thishypovascular lesion. A surgical procedure performed several weeks laterconfirmed that this lesion was colon carcinoma. Arrows indicate lesionsites.

FIG. 6 illustrates gamma camera imaging results at 6 days using ¹³¹ Ilabeled AS 33. ¹³¹ I-AS 33 scan demonstrated a large mass in the rightlobe of the liver. This was also identified on CTT. A smaller lesion isseen in the left lobe of the lesion which was not identified on CTT scanbut was confirmed at surgery by biopsy. Arrows indicate lesion sites.

FIG. 7 summarizes tissue and blood dosimetry results for 1 patientinfected with control antibody ¹³¹ I-TA99, specific antibody ¹²⁵ I-AS 33and ^(99m) Tc HSA (for blood flow analysis). The large differences intissue (tumor/liver) and blood (tumor/plasma) uptake ratios between AS33 and control antibody demonstrate that localization is specific.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of imaging a colorectal carcinomalesion in a human patient which comprises administering to the patient amonoclonal antibody capable of binding to a cell surface antigenassociated with the colorectal carcinoma lesion and which is labeledwith an imaging agent under conditions so as to form a complex betweenthe monoclonal antibody and the cell surface antigen, imaging anycomplex so formed, and thereby imaging the colorectal carcinoma lesion.

This method is useful in imaging primary colorectal carcinoma lesionsand metastatic colorectal carcinoma lesions.

The monoclonal antibody designated AS 33 (A33) is a monoclonal antibodywhich is capable of binding to a cell surface antigen which isassociated with colorectal carcinoma lesions and may be administered inan amount from about 0.01 mg to about 100 mg although an amount ofmonoclonal antibody from about 0.2 mg to about 50 mg is preferred. Themonoclonal antibody of the invention may be administered by intravenousinfusion.

This invention further provides a method of imaging a colorectalcarcinoma lesion in a human patient wherein the monoclonal antibody AS33 (HB 8779) is labeled with an imaging agent, such as ¹³¹ I, andadministered to the patient to bind to a cell surface antigen associatedwith a colorectal carcinoma lesion so as to form a complex between themonoclonal antibody AS 33 and the cell surface antigen, and therebyimaging colorectal carcinoma lesions.

This invention also provides the monoclonal antibody AS 33 labeled witha radioisotope imaging agent.

This invention further provides the monoclonal antibody AS 33 labeledwith the imaging agent ¹³¹ I.

This invention also provides a method of imaging a colorectal carcinomalesion wherein the monoclonal antibody AS 33 is labeled with ¹³¹ I andthe amount of ¹³¹ I -AS 33 administered to the patient is from about 0.1mCi to about 50.0 mCi, although an amount of ¹³¹ I from about 2.0 mCi toabout 5.0 mCi is preferred.

This invention also provides a method of detecting a colorectalcarcinoma lesion wherein the monoclonal antibody AS 33 labeled with animaging agent is administered in an amount from about 0.01 mg to about100 mg, although an amount of about 0.2 mg to about 50 mg is preferred.

Imaging of the complex comprising the labeled monoclonal antibody AS 33(HB 8779) and the cell surface antigen associated with a colorectalcarcinoma lesion may be carried out using a gamma camera or using singlephoton emission computed tomography.

This invention also provides a monoclonal antibody AS 33 (ATCC Accessionnumber HB 8779) and the hybridoma cell line which produces it.

This invention also provides the monoclonal antibody AS 33 (HB 8779) andthe hybridoma all line which produces it.

This invention further provides monoclonal antibody labeled with animaging agent. The imaging agent may comprise the radioisotope ¹³¹ I.

This invention also provides a method of imaging a colorectal carcinomalesion wherein the amount of ¹³¹ I labeled AS 33 administered is in anamount from about 0.1 mCi to about 50.0 mCi, although an amount fromabout 2.0 mCi to about 5.0 mCi is preferred.

This invention further provides a composition comprising an effectiveimaging amount of the monoclonal antibody AS 33 labeled with an imagingagent and a pharmaceutically acceptable carrier.

MATERIALS AND METHOD

Tissue Culture: Cultured human colon cancer cell lines were obtainedfrom the Human Cancer Immunology Tumor Bank (Sloan-Kettering Institute,New York). Cultures were maintained as reported previously (2).

Production of Mouse mAb: BALB/c mice were immunized with (culturedpancreas carcinoma cells (ASPC-1). Subcutaneous and intraperitonealinjections of 1×10⁷ cells were given three to ten times at intervals of2 weeks. Three days after the last injection immune spleen cells werefused with mouse myeloma MOPC-21 NS/1 cells as described (3). Culturesupernatants were tested for reactivity with a panel of cell linesderived from colon cancer and other tumor types using the anti-mouse Igmixed hemabsorption assay (MHA) or protein A assay (PA). After three ormore steps of subcloning, hybridoma cells were injected subcutaneouslyinto nu/nu mice, (Swiss background) and sera from tumor-bearing micewere collected and used for serological, immunopathological andbiochemical studies. In general, these methods have been describedpreviously (2, 3).

Serological Procedures: The MHA assay for detecting antigens on culturedcells using rabbit anti-mouse IG and mouse anti-sheep red blood cellshas been described (2, 3). Absorption tests, assessment of heatstability, proteinase sensitivity and antibody subclass determinationwere also performed as described (2-5). The reactivity of the antibodieswith short term cultures of colonic tumors and normal colonic tissueswas performed as described previously in co-pending patent applicationU.S. Ser. No. 118,411 (now abandoned), filed Nov. 6, 1987, continuationof U.S. Ser. No. 724,991, filed Apr. 19, 1985 (now abandoned).

Immunohistochemistry: Fresh frozen tissue sections were assayed forreactivity with mouse mAbs using fluorescein-conjugated goat anti-mouseIg (Cappel Laboratories, Cochranville, Pa.) as described (6)Immunoperoxidase staining, using mAbs and peroxidase-conjugated goatanti-mouse Ig and 3-amino-9-ethylcarbazol (Histoset, Ortho DiagnosticSystem) was carried out following procedures recommended by themanufacturer.

Monoclonal AS 33 Ascites Production

General procedures: Nude mice are from MSKCC colony. Mice are purchasedfrom The Jackson Laboratory, Bar Harbor, Me. Animal rooms are monitoredfor the presence of viruses. Cells and ascites must be bacteria-,fungus-, and myco-plasma-free. Ascites are screened for murine virusesbefore purification. Sterile procedures are used throughout productionand purification procedures. A test for sodium azide is performed onchromatography columns prior to use. Quality and safety tests areperformed on all final products.

Ascites production

1. Female nu/nu mice (2-3), primed with pristane oil, are injectedintraperitoneally with 3-5×10⁶ hybridoma cells (Passage 1).

2. When tumor is established, individual ascites are collected frominjected mice, and antibody titers determined. Ascites cells are frozenand inoculated intraperitoneally into pristane-primed, irradiated maleor female C3^(y) B⁶ F¹ hybrid mice (Passage 2).

3. Passage 2 ascites cells are injected into 75-100 pristane-primed,irradiated F¹ male or female mice (passage 3). Donor cells are pooledaccording to donor's titer; cells not needed for injection are storedfrozen.

4. The same procedure is repeated with Passage 4 ascites cells, andcontinued serially until antibody titers decrease.

5. Backup frozen cells and 3 and 4 are used to increase the ascitespool, and frozen cells from Passages 1 and 2 are used to institute theprocedure.

6. A final batch of 2-3 liters is established, the antibody titerdetermined, and a sample tested for contamination with murine viruses.

METHOD FOR PURIFICATION OF MONOCLONAL ANTIBODY AS 33

Buffers:

Buffer A--0.03 M HEPES/NaOH, PH 8.0

Buffer B--0.5 M HEPES/NaOH, pH 8.0

Buffer C--0.03 M HEPES/NaOH, pH 8.0

Buffer D--0.05 M Phosphate/NaOH, 0.15 M NaCl, pH 7.0

Saturated Ammonium Sulfate

The buffers are prepared with water for injection, USP and filteredthrough 0.22 uM filters. No preservatives are used in any of the bufferswith the exception of those buffers used for column storage, to whichsodium azide is added to a final concentration of 0.02%.

All glassware, tubing and other apparatus are steam, dry heat or gassterilized. Unless otherwise stated, all procedures are carried out at2° C. to 8° C.

Procedure

Lipoproteins are removed from ascites by centrifugation at 135,000×G for3 hours, and the material is stored in quantities suitable forpurification.

Antibody is purified using the following steps:

1. Ammonium sulfate precipitation 0%-45%; pH is maintained at pH 7.4 10NaOH.

2. After equilibration of for 60 min., the suspension is pelleted bycentrifugation at 10,000×G for 15 min.

3. The pellet is dissolved in a small volume of Buffer A and centrifugedat 2000×G.

4. The supernatant from step 3 is applied to a Sephadex G50 superfinecolumn (5×42 cm), equilibrated with Buffer A, at a flow rate of 25ml/min at room temperature.

5. The eluted protein fraction is applied to a S-Sepharose Fast FlowColumn (5×10 cm), equilibrated with Buffer A, at a flow rate of 35ml/min at room temperature.

6. After the protein elution curve returns to baseline, the runningbuffer is changed to 93% Buffer A and 7% Buffer B.

7. After the protein elution curve returns to baseline, the runningbuffer is changed to 93% Buffer A and 7% Buffer B.

8. The protein eluted after running 100% Buffer C is applied to aSephadex G50 superfine column (5×42 cm), equilibrated with buffer D, ata flow rate of 25 ml/min.

9. The eluted protein is sterilized by filtration, vialed and frozen.This is the A33 final product.

EXPERIMENTAL RESULTS

From 23 fusions of immune splenocytes with NS/1 myeloma cells, 4antibody-producing clones with a restricted pattern of reactivity withcultured cell lines were selected for detailed analysis. Theseantibodies were found to be unreactive with A, B, H, Le^(a), Le^(b), X,Y and I specific glycoproteins. The serological specificity of theseantibodies was determined on a panel of 154 established human cancercell lines, on 10 short-term cultures of human fibroblast and kidneyepithelial cells and on a panel of short-term cultures of normal colonbenign and malignant colon tumors. The reactivity of these antibodieswith a panel of normal human fetal and adult tissues, as well as normalcolon epithelium and colon cancer tissues from 55 patients, was examinedusing indirect immunofluororescence and the ABC immunoperoxidasetechniques.

AA 33

Monoclonal antibody AS 33 reacted with 5/17 colon carcinoma and 1/3pancreatic carcinoma cell lines with a titer of 10³ -10⁶ AS 33 was alsoreactive with 3/5 T leukemia cell lines but all other tumor cell typestested were unreactive (See FIG. 1).

Reactivity of T-cell leukemias with monoclonal antibody AS 33 did notcorrelate with expression of other T-cell related antigens, includingthose detected by OKT4, OKT6, OKT11, T37, CL3-3 and CL3-40.

In tissues, reactivity of monoclonal antibody AS 33 was restricted tocarcinoma tissue and normal colonic mucosa in the majority of cases ofcolon cancer studied (50/59) including primary tumors and livermetastases. AS 33 reactivity was observed in all cases in 90 to 100% oftumor cells. Reactivity was strong and homogenous.

AS 33 also showed a very strong cytoplasmic reactivity in all samples ofnormal colonic mucosa, including samples of ascending, transverse anddescending colon. Other normal tissues which showed AS 33 antigenexpression were the ductiles of the major salivary glands and positivebut heterogenous reaction in the ducts and acini of the normal breast(See FIG. 2).

Other normal tissues tested including liver, kidney, lung, stomach,bladder, cervix uteri, ovary, larynx and adrenal gland were negative(See FIG. 2).

Other carcinomas tested showed ± reactivity in an infiltrating ductcarcinoma of breast scattered positive cells were seen in a renal cellcarcinoma and 4 gastric carcinomas for AS 33.

The antigen detected by monoclonal antibody AS 33 was not destroyed byheating at 100° C. for 5 minutes and it was weakly detectable incholoroform/methanol extracts of ASPC-1 cells.

Imaging Experiments

The monoclonal antibody AS 33 can be labeled with ¹³¹ I by thechloramine-T method and retains 50-70% of its immunoreactivity as testedby sequential quantitative absorption assays.

Labeling of Purified AS 33 with ¹³¹ I

Sterile technique and pyrogen-free glassware were used in all labelingsteps. About 0.2 to about 25.0 mg of AS 33 is added to 0.5 ml of 0.15 MNaCl/0.05 M H₃ PO₄, pH 7.5. The antibody is iodinated with 5 mCi ¹³¹ Ias NaI and 50 microliters of chloramine-T (10 mg/ml) for 30 sec at 24°C. The reaction is stopped by addition of 50 mCi sodium metabisulfate(12 mg/ml) and transferred to a 25 ml Biogel P30 column (50-100 mesh).Fractions with the highest amount of radioactivity are pooled andfiltered through a 0.2 micron filter. Using similar techniques, AS 33also was labeled with 2 mCi ¹²⁵ I for use in six patients who receivedboth mAbs, AS 33 and control TA99. Monoclonal antibody TA99 was labeledas described above with 5 mCi ¹³¹ I.

Administration of .sup. 131 I AS 33

The uptake of ¹³¹ I in the thyroid was blocked by Lugol's solution; 5drops po bid starting 24 h prior to antibody administration. ¹³¹ I AS 33was administered in doses of about: 0.2 mg; 2 mg; 10 mg; and 25, by onehour intravenous infusion in about 100 to about 200 ml of saline/5%human serum albumin.

Fifteen patients, all with liver metastases and were scheduled toundergo surgery were administered ¹³¹ I labeled AS 33. ¹³¹ I-AS 33 wasadministered intravenously at 5 dose levels (0.2 mg, 2 mg, 10 mg, 25 mgand 50 mg), which was labeled with about 2.0 to about 5.0 mCi of ¹³¹ I.Three patients were entered at each dose level.

Imaging Results

Evaluation included whole body imaging of ¹³¹ I-AS 33 using a gammacamera (daily, day 1-8); blood flow study using ^(99m) Tc-albuminliver/spleen scan, CTT scan, hepatic arteriogram (once during the weekof imaging); blood pharmacokinetics (day 1-8); a biopsy of tumors andnormal tissues (day 8). Fifteen patients were entered, and 12 patientswere evaluated. Antibody localization to tumor was documented byexternal imaging and radioactive tissue counting in all but one patient.Examination of the resected tumor specimens revealed that most of theradioisotope was localized in the rim of viable tumor tissue surroundingthe large necrotic centers typical for these metastatic lesions. Themaximal tumor: liver ratio ranged from 6.9.1 to 100.1:1, except for thepatient whose liver metastases could not be visualized by externalimaging; in this case the ratio was 2.7:1. In three patients with extrahepatic sites of disease (primary/recurrent colon tumors, lymph nodemetastases or lung metastases) antibody localization to tumor tissue wasalso seen. At the highest dose level administered so far (25 mg),imaging of liver metastases or lung metastases) antibody localization totumor tissue was also seen. Also at the highest does level administeredso far (25 mg), imaging of liver metastases was more distinct that atlower dose levels. This difference is not explained by retention of ahigher proportion of the injected does in tumor tissue which was 0.9-15.0×10-³ per gram over the entire dose range. Rather, blood clearanceof ¹³¹ I-AS 33 was found to be more rapid at the highest dose level,resulting in tumor: plasma ratio of 13.0-17.8 on day 8 in the threepatients receiving the 25 mg dose as opposed to ratios of 4.1 to 11.3 inpatients treated at the lower does levels.

FIG. 3 summarizes tissue and blood dosimetry data collected from thetwelve patients at day 7-8 who were administered ¹³¹ I labeled AS 33.

Other imaging agents which are well known to those skilled in the artare useful in the practice of this invention. Other such agents include,but are not limited to the radioisotopes: ¹²⁴ I; ¹²³ I; ⁹⁹ Tc; and ¹¹¹In.

Additionally, other methods of imaging the complex comprisingradiolabeled antibody and cell surface antigen associated withcolorectal carcinoma may be used. Other such methods include, but arenot limited to single photon emission computed tomography (SPECT) andpositron emission tomograph (PET).

The method of this invention also may be used as a therapy in thetreatment of colorectal carcinomas, i.e., as a method of deliveringradioisotopes to the colorectal carcinoma lesion.

The hybridomas which produce the monoclonal antibodies designated AS 33and TA99 have been deposited with the American Type Culture Collectionin Rockville, Md., U.S.A. 20852, and identified by ATCC Accession Nos.HB 8779 and HB 8704, respectively. These deposits were made pursuant tothe provisions of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms For The Purposes of Procedure (BudapestTreaty).

What is claimed is:
 1. The monoclonal antibody AS 33 (HB 8779).
 2. The hybridoma cell line which produces the monoclonal antibody of claim
 1. 3. The monoclonal antibody of claim 1, labeled with an imaging agent.
 4. The monoclonal antibody of claim 3, wherein the imaging agent comprises a radioisotope imaging agent.
 5. The monoclonal antibody of claim 4, wherein the radioisotope comprises ¹³¹ I.
 6. The monoclonal antibody of claim 5, wherein the amount of ¹³¹ I is from about 0.1 mCi to about 50.0 mCi.
 7. The monoclonal antibody of claim 6, wherein the amount of ¹³¹ I is from about 2.0 mCi to about 5.0 mCi.
 8. A composition comprising an effective imaging amount of the monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier. 